The present study was planned to investigate the disposition kinetics of levofloxacin in plasma of female native Barky breed sheep after single intravenous (IV) and intramuscular (IM) administration of 4 mg/kg body weight. The concentrations of levofloxacin in the plasma were measured using high-performance liquid chromatography (HPLC) with a UV detector on samples collected at 0, 0.08, 0.16, 0.33, 0.5, 1, 2, 4, 6, 8, 10, 12, 18, 24, 32, and 48 h after treatment. Following intravenous injection, the decline in plasma drug concentration was biexponential with half-lives of (t(1/2α)) 0.33 ± 0.12 h and (t(1/2β)) 3.29 ± 0.23 h for distribution and elimination phases, respectively. The volume of distribution at steady state V((d(ss))) was 0.86 ± 0.23 l/kg. After intramuscular administration of levofloxacin at the same dose, the peak plasma concentration (C(max)) was 3.1 ± 0.35 μg/mL and was obtained at 1.64 ± 0.29 h (T(max)), the elimination half-life (T(1/2el)) was 3.58 ± 0.30 h, and AUC was 20.24 ± 1.31 μg.h/mL. The systemic bioavailability was 91.35 ± 6.81 %. In vitro plasma protein binding was 23.74%. When approved therapy fails, levofloxacin may be used in some countries for therapy of food animals, however, that is not true in the US.